The short-term goal of this project is to understand the trafficking and activation of pertussis toxin (PT) within mammalian cells. Bordetella pertussis colonizes the human respiratory tract and causes the disease pertussis (whooping cough). Several systemic symptoms accompany this disease, even though B. pertussis is not thought to invade beyond the respiratory tract. Instead these symptoms are thought to be due to the action of PT, an exotoxin produced by B. pertussis. PT is an ADP ribosyltransferase that modifies several heterotrimeric G proteins, causing a wide range of effects on signaling in mammalian cells. How PT is transported within mammalian cells to arrive at its target proteins in an active form is largely unknown. In addition, how PT is transported from the respiratory tract to systemic sites is completely unknown. Understanding the mechanisms utilized by this complex toxin to achieve these effects will provide key information on the cell biology of PT and will help to provide a groundwork for studies to elucidate the role of this toxin in B. pertussis infection and disease. In addition, this information may allow development of therapeutics to combat the effects of the toxin and may also allow improvement of existing pertussis vaccines that include PT or development of novel vaccine molecules using PT as a intracellular delivery vector. We have preliminary data indicating that (i) PT may undergo retrograde intracellular trafficking through the Golgi apparatus and endoplasmic reticulum (ER) en route to its cytosolic target proteins, (ii) that proteolytic processing of the active Si subunit of cell-associated PT occurs and may be important for its activity, and (iii) that there is apparent transcytosis of active PT across intact polarized epithelial cells in culture. Therefore the specific aims of this proposal are to investigate the trafficking, processing and transcytosis of PT in mammalian cells and the key features of this toxin that mediate these events.